To understand the molecular basis of developmental control of cell division during C. elegans organogenesis, two different approaches were taken. First, a screen was performed to identify mutants with altered numbers of intestinal nuclei using a reporter transgene specific to the intestinal nuclei. The intestine displays three different cell division patterns; mitosis, karyokinesis and endoreplication, therefore, in this screen we could potentially isolate mutants in genes affecting any of these different cell cycles. An F2 semi-clonal screen was performed and mutants with fewer or supernumerary numbers of intestinal nuclei were isolated. One mutant, rr31, with twice the wild type complement of intestinal nuclei was mapped and the defect was subsequently shown to be due to a gain-of-function mutation in the cell cycle phosphatase cdc-25.1. Further characterization of the cdc-25.1(gf) mutant, showed that the extra intestinal cells arise from an additional division of the intestinal cell precursors during embryogenesis, and that this phenotype is unique to the intestinal lineage. (Abstract shortened by UMI.)

No description available.

In Caenorhabditis elegans, mutations in the clk-1 gene result in delayed embryonic and post-embryonic development, a slowing down of rhythmic behaviors and an extended life span. CLK-1 encodes the demethoxyubiquinone (DMQ or DMQn) hydroxylase in the ubiquinone (CoQ or Qn) biosynthesis pathway. Thus, clk-1 mutants produce DMQ instead of CoQ. In order to understand the relationship between the CoQ biosynthesis defect and the pleiotropic phenotype of clk-1 mutants, I isolated a deletion mutant, coq-4(qm143), in C. elegans. In Saccharomyces cerevisiae, mutants in COQ4, the coq-4 homologue, do not produce ubiquinone, like those in COQ7, the clk-1 homologue. coq-4(qm143) is a non-strict maternal-effect lethal mutation. Most of the progeny from a homozygous coq-4(qm143) hermaphrodite die during embryogenesis. However, homozygous coq-4(qm143) hermaphrodites from a heterozygous mother can develop and behave normally until adulthood. As adults, they become uncoordinated and paralytic, and are defective in egg-laying. Unlike hermaphrodites, homozygous coq-4(qm143) males are fully maternally rescued. The qm143 is a 1469 base pair deletion, which completely removes the coq-4 gene and does not affect the coding sequence of any other gene. By performing germline transformation, I also showed that the non-viable phenotype of coq-4 (qm143) is indeed due to the removal of the coq-4 gene itself. The preliminary study of COQ-4 expression pattern by using a COQ-4::GFP fusion protein indicates that COQ-4 is expressed in mitochondria of the worm.

Rapid progress has been made toward understanding the significance of CDK inhibitor proteins (CKIs) in the regulation of cell cycle progression. The overall goal of this study has been targeted to further expand our knowledge of CKI function through the investigation of a previously uncharacterized CKI named cki-2 during development in C. elegans. The characterization of cki-2 using a reverse genetic approach called co-suppression has revealed a novel mechanism that cki-2 and its related cell cycle regulators are required for the appropriate elimination of centrioles during oogenesis. Loss of cki-2 in the germ line caused perdurance of centrioles into the one-cell embryo, resulting in supernumerary centrosomes and aberrant cell divisions in the first cell cycle. This was significantly suppressed by reduction of cyclin E and a Cdk2 homologue, indicating that these cell cycle regulators are involved in this critical developmental process. In order to further understand the function of cki-2, a yeast two-hybrid screen was conducted which allowed us to identify three CKI-2 interacting proteins: orthologues of PCNA (PCN-1), SUMO (SMO-1), and a RING finger protein called RNF-1. CKI-2 has functionally separable domains in its amino (Cyclin/Cdk binding)- and carboxy (PCNA binding)-terminus and they exert distinct roles in cell cycle progression. It was observed that CKI-2 is covalently modified by SUMO on its N-terminus and this causes CKI-2 to relocalize to thr nucleolus, which is associated with its rapid degradation. Since many RING finger proteins act as components of the multi-subunit E3 ubquitin ligases, we speculated that RNF-1 might be involved in the CKI-2 degradation. This possibility was tested by co-expression of RNF-1 with CKI-2, revealing that co-expression of RNF-1 suppresses the embryonic lethality caused by the CKI-2 overexpression and moreover, this is correlated with an increased rate of CKI-2 degradation. In addition, western blot analyses performed on different genetic backgrounds suggested that the CKI-2 degradation occurs in an ubiquitin-dependent manner through the proteasome-mediated proteolysis pathway. Furthermore, a yeast-based assay developed to test a possible role of SUMO in modulating the CKI-2/RNF-1 interaction demonstrated that SUMO may antagonize the interaction between CKI-2 and RNF-l, these highlighting an intriguing model that appropriate levels of CKI-2 are regulated through ubiquitin-dependent proteolysis mediated by RNF-l, and which maybe modulated by SUMO.

No description available.

No description available.

Initiation of postembryonic development is an important event for normal C. elegans development. Extrinsic factors affect development as well as intrinsic developmental cues. In order to investigate the molecular basis of initiation of postembryonic development, a genetic screen was performed to identify temperature-sensitive mutants that cannot initiate the cell divisions associated with postembryonic development at the restrictive temperature. Hydroxyurea (HU), a DNA replication inhibitor, was used as a tool to select against worms that initiate postembryonic cell divisions and/or the developmental program. 1,600,000 haploid genomes were screened, and 20 mutants have been isolated. 6 of them have been mapped to a relatively small genetic interval, and one inx-6 has been cloned and encodes an innexin family protein. Mutation of inx-6 caused abnormalities in pharyngeal pumping, resulting in worms that could not feed. The functions of a cyclin B homologue (ZC168.4) in postembryonic development have also been studied since cyclin B mutants also have postembryonic developmental arrest phenotype. Results indicate that zygotic expression of cyclin B is absolutely required for normal postembryonic development. Moreover, we found a novel function of this cyclin B homologue, which demonstrates an uncommon paternal effect required for spermatogenesis and/or fertilization.

No description available.